Synthesis of MOF-5/polyethersulfone (PES) mixed matrix membranes for enhancing membrane filtration performance in polyphenol purification.

The various apple products industries produce a large amount of apple residue, which is easily fermented, causes environmental pollution, and its disposal cost is high, but is rich in nutrients, such as polyphenols. Polyphenols can be purified to realize high-value deep processing of apple pomace and to promote energy reuse of food waste. In this study, the highly selective purification of polyphenols was achieved by membrane filtration using prepared Metal-organic framework (MOF)-5/PES mixed matrix membranes with apple peels as raw material. The polyethersulfone mixed matrix membrane was loaded with MOF-5 by the phase inversion method, and their structural and physicochemical properties were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). Zeta potential and specific surface area of MOF-5 particles were measured, as well as the water contact angle and anti-fouling properties of the mixed matrix membrane were analyzed. It was confirmed that the membrane loaded with MOF-5 showed better hydrophilicity and mechanical properties compared with the pristine polyether sulfone membrane. Under practical conditions, the increased hydrophilicity could enhance the anti-fouling properties of membranes, which would improve the flux recovery ratio of membranes. In addition, the prepared MOF-5/PES mixed matrix membrane was applied to the purification of polyphenols, showing excellent purification performance of polyphenols. In particular, the purity of polyphenol after membrane filtration could reach 70.45% when the additional amount of MOF-5 was 10%. This research provides a method to prepare MOF-5/PES mixed matrix membranes, which effectively solves the problem of unstable and unsatisfactory purification effect of commercially available membranes, promotes the development of new materials in membrane science, and realizes high-value deep processing and comprehensive resource development of food waste using membrane filtration.

Synthesis, catalysts and enhancement technologies of biodiesel from oil feedstock - A review.

Biodiesel is considered as one of the most promising alternative fuels due to the depletion of fossil fuels and the need to cope with potential energy shortages in the future. This article provides a thorough analysis of biodiesel synthesis, covering a variety of topics including oil feedstock, synthesis methods, catalysts, and enhancement technologies. Different oil feedstock for the synthesis of biodiesel is compared in the review, including edible plant oil, non-edible plant oil, waste cooking oil, animal fat, microbial oil, and algae oil. In addition, different methods for the synthesis of biodiesel are discussed, including direct use, blending, thermal cracking, microemulsions, and transesterification processes, highlighting their respective advantages and disadvantages. Among them, the transesterification method is the most commonly used and a thorough examination is given of the benefits and drawbacks of utilizing enzymatic, heterogeneous, and homogeneous catalysts in this process. Moreover, this article provides an overview of emerging intensification technologies, such as ultrasonic and microwave-assisted, electrolysis, reactive distillation, and microreactors. The benefits and limitations of these emerging technologies are also reviewed. The contribution of this article is offering a thorough and detailed review of biodiesel production technologies, focusing mainly on recent advances in enhanced chemical reaction processes. This provides a resource for researchers to assess and compare the latest advancements in their investigations. It also opens up the potential for enhancing the value of oil feedstocks efficiently, contributing to the development of new energy sources.

Folic Acid Protects Against Kidney Damage in Mice with Diabetic Nephropathy by Inhibiting M1 Macrophage Polarization via Nuclear Factor-k-gene Binding Pathway.

Kidney damage is one of the most common complications of diabetes, and inflammation caused by macrophage infiltration plays an important role. Folic acid (FA), a water-soluble vitamin, was previously found to affect inflammation by regulating macrophage polarization. In our study, we aimed to investigate the effect of FA on renal injury in mice with diabetic nephropathy (DN). We found that FA treatment ameliorated diabetic metabolic parameters in mice with DN, including reducing 24-hour food consumption, 24-hour urine volume and 24-hour water intake and increasing body weight and serum insulin. Of note, FA treatment improved renal functional and structural damage in mice with DN. In addition, FA treatment significantly reduced the number of renal infiltrating M1 macrophages, inflammatory cytokine FA stimulation significantly reduced the increase in F4/80+CD86+ cell ratio, inflammatory factor content and p-p65/p65 protein expression induced by high glucose exposure in RAW264.7 cells. All in all, our results indicated that FA protects against kidney damage in mice with DN by inhibiting M1 macrophage polarization, and its mechanism may be related to the inhibition of nuclear factor-k-gene binding (NF-kB) signaling pathway.

A review of the polyphenols purification from apple products.

Apple polyphenols are one of the major bioactive compounds in apple products and have strong anti-inflammatory effects and the ability to prevent chronic diseases with health benefits. The development of apple polyphenol products is dependent on the extraction, purification and identification of apple polyphenols. The extracted polyphenols need to be further purified to improve the concentration of the extracted polyphenols. This review, therefore, presents the studies on the conventional and novel methods for polyphenols purification from apple products. The different chromatography methods, as one of the most widely used conventional purification methods, for polyphenol purification from various apple products are introduced. In addition, the perspective of the adsorption-desorption process and membrane filtration technique in enhancing the purification of polyphenols from apple products are presented in this review. The advantages and disadvantages of these purification techniques are also discussed and compared in depth. However, each of the reviewed technologies has some disadvantages that need to be overcome, and some mechanisms need to be further identified. Therefore, more competitive polyphenols purification techniques need to emerge in the future. It is hoped that this review can provide a research basis for the efficient purification of apple polyphenols, which can facilitate their application in various fields.

A review of the polyphenols extraction from apple pomace: novel technologies and techniques of cell disintegration.

Apple pomace, a solid waste produced during industrial processing of apple juice or cider, is a rich source of high value-added compounds such as polyphenols. This review summarizes present studies on the qualitative and quantitative methods, including Folin-Ciocalteu colorimetric, high pressure liquid chromatography (HPLC) and fluorescence spectrum, as well as enhanced extraction methods of polyphenols in apple pomace by different traditional and novel technologies, including ultrasounds (US), microwave (MW), pulsed electric fields (PEF), high voltage electrical discharges (HVED) and enzyme. The principles and characteristics of different effective enhanced extraction technologies of polyphenols in apple pomace were compared. In addition, the different cell disruption analysis methods, such as destructive detection method (electrical conductivity disintegration index, Zc), image analysis method (including scanning electron microscopy, SEM, and confocal laser scanning microscopy, CLSM), and nondestructive method (such as magnetic resonance imaging, MRI) are presented in this review. The study proved that there was a correlation between destructive detection method and image analysis method. However, each of the technologies reviewed in this study has some disadvantages to overcome, and some mechanisms need to be further substantiated. Therefore, more competitive techniques for polyphenols extraction and analysis of cell disintegration are needed to emerge in the future.

Roles of Nrf2/HO-1 and HIF-1α/VEGF in lung tissue injury and repair following cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury (CIRI) leads to injury in distant organs, most commonly the lungs, although limited studies have examined self-protective mechanisms during CIRI-induced lung injury. Here, we investigated self-protective mechanisms that attenuate stress-related injury and promote the angiogenetic repair of epithelial function during CIRI-induced lung injury by measuring nuclear factor erythroid-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF-1α) levels. A CIRI model was established in male Sprague-Dawley rats by blocking the middle cerebral artery. Rats were divided into five subgroups based on the reperfusion time (6, 12, 24, 48, and 72 hr). Lung injury was assessed using a semiquantitative score and a thiobarbituric acid-based method of determining malonaldehyde production. Lung tissue angiogenesis was detected by CD34 and CD31 immunolabeling. Changes in Nrf2, heme oxygenase-1 (HO-1), HIF-1α, vascular-endothelial growth factor (VEGF), phosphatidylinositol 3-kinase (PI3K), extracellular-regulated kinase1/2 (ERK1/2), and phospho-ERK1/2 ( p-ERK1/2) protein- and mRNA-expression levels were measured by immunohistochemistry and reverse transcription polymerase chain reactions, respectively. Oxidative stress induced by cerebral ischemia/reperfusion (CI/R) caused lung injury. Expression of the Nrf2/HO-1 antioxidative stress pathway in lung tissues increased following CI/R, peaking after 24 hr. PI3K, ERK, and p-ERK1/2, which act upstream of Nrf2/HO-1, were expressed at higher levels in the CI/R-model group, consistent with the general trends observed for Nrf2/HO-1. Within 72 hr post-CI/R, HIF-1α, and VEGF expression significantly increased versus the sham group. Thus, during CIRI-induced lung injury, the body may upregulate antioxidative stress activities and promote angiogenesis to repair the endothelial barrier through the Nrf2/HO-1 and HIF-1α/VEGF signaling pathways, enabling self-protection.

Analyses of clinical efficacy of ultrasound-guided radiofrequency ablation in liver cancer adjacent to the gallbladder and its prognosis.

PURPOSE: To explore the clinical efficacy of ultrasound-guided radiofrequency ablation (RFA) in liver cancer adjacent to the gallbladder and to analyze its prognosis. METHODS: 80 patients with liver cancer adjacent to the gallbladder, who were admitted to our hospital from January 2015 to April 2018, were enrolled and divided into the Observation group (n=40) and the Control group (n=40). All of the patients underwent cholecystectomy and lymph node dissection combined with postoperative chemotherapy. RFA was performed in the Observation group, while radical cholecystectomy and radical hepatectomy were conducted simultaneously in the Control group. Follow up was by telephone, and tumor-associated factor levels, liver function and cellular and humoral immune function-related indicators at 1 month after intervention, tumor size before and after treatment and cases of normal alpha-fetoprotein (AFP) level and tumor disappearance after treatment were compared between the two groups. The complications rates during treatment (increase in transaminases, elevation of bilirubin, intratumoral hemorrhage, bile duct injury and gastrointestinal perforation), clinical efficacy and 1-year survival in the two groups were statistically analyzed. RESULTS: At 1 month after intervention, the Observation group had substantially lower levels of tumor-associated factors AFP, carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) (p<0.05), obviously lower levels of liver function indicators aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), indirect bilirubin (IBIL) and direct bilirubin (DBIL) (p<0.05), but distinctly higher levels of immunoglobulin G (IgG), IgA and IgM, cluster of differentiation 4+ (CD4+), CD8+ and CD4+/CD8+ (p<0.05) than the Control group. Before and after treatment, the tumor size in the Observation group was smaller than in the Control group (p<0.05). The Observation group exhibited notably more cases of normal APF level and tumor disappearance after treatment (p<0.05), markedly lower incidence rates of increase in transaminases, elevation of bilirubin, intratumoral hemorrhage, bile duct injury and gastrointestinal perforation during treatment (p<0.05) than the Control group. Additionally, the rate of stable disease (SD) was notably higher and the 1-year survival rate was higher in the Observation group than in the Control group (p<0.05). CONCLUSIONS: RFA for liver cancer adjacent to the gallbladder can effectively lower the levels of tumor markers, improve liver function and enhance immunity, with a few operative complications and high efficacy, so it has a positive impact in prolonging the survival of patients.

Cerebral ischemia/reperfusion injury induces airway mucus hypersecretion in rats and activates IL­13­associated inflammatory mechanisms.

The majority of patients that suffer a stroke have excessive sputum, which accelerates the development of pulmonary complications. However, it is unclear whether cerebral ischemia and reperfusion (I/R) injury induces mucus hypersecretion, and the potential role of inflammation remains unknown. In the present study, the reversible middle cerebral artery occlusion model was applied in rats to induce cerebral I/R injury. The rats were grouped according to the duration of reperfusion (6, 12, 24, 48 and 72 h). Neurological dysfunction was evaluated by Longa scoring and lung dry­to­wet weight (dw/ww) ratios were determined to reflect the degree of mucus secretion. Inflammatory factor interleukin­13 (IL­13) and tumor necrosis factor­α (TNF­α) levels in serum and bronchoalveolar lavage fluid (BALF) were determined by enzyme­linked immunosorbent assay. Pulmonary levels of mucin 5AC (MUC5AC) and key molecules involved in nuclear factor­κB (NF­κB) signaling were determined by western blotting and immunohistochemistry. Rats with cerebral I/R had impaired neurological function, which was associated with the length of reperfusion time. In addition, the dw/ww lung ratio decreased and the pulmonary expression of MUC5AC increased with the increase in severity of neurological dysfunction, indicating that cerebral I/R may induce mucus hypersecretion in a reperfusion time­dependent manner. IL­13 and TNF­α levels in serum and BALF, as well as the nuclear translocation of NF­κB p65 in pulmonary tissues, significantly increased following cerebral I/R, which suggests that the activation of IL­13 and NF­κB inflammatory pathways may be involved. The present study concluded that cerebral I/R injury may induce airway mucus hypersecretion by activating IL­13 and NF­κB inflammatory pathways.

Functional single-walled carbon nanotubes 'CAR' for targeting dopamine delivery into the brain of parkinsonian mice.

Current treatments for Parkinson's disease (PD) are limited, partly due to the difficulties posed by the blood brain barrier (BBB) when delivering drugs to the brain. Herein, we explore the feasibility and efficacy of functional single-walled carbon nanotubes 'CAR' (SWCNT-PEGs-Lf) which carry and target-deliver dopamine (DA) to the brain in PD mice for treatment. SWCNTs can penetrate the cell-membrane remarkably, with the characteristics including high drug-loading and pH-dependent therapeutic unloading capacities. It has been reported that polyethylene glycol (PEG)-coated SWCNTs could increase the circulation time and thus prolong the concentration gradient of SWCNTs to the brain. Besides, an obvious lactoferrin-nanoparticle (Lf-NP) accumulation in the striatum, wherein the pharmacological target site of PD has been reported, a dual modification of PEG and Lf onto SWCNTs was applied and thus a specific 'CAR' to carry DA. The results from in vitro studies demonstrate that with 20 mol L-1 DA loaded onto SWCNT-polyethylene glycol (PEGs) in addition to 100 µmol L-1 6-hydroxydopamine (6-OHDA), the activity of PC12 cells increases significantly (p < 0.05), and that the lactate dehydrogenase (LDH) levels and reactive oxygen species (ROS) content also significantly decrease (p < 0.01). Furthermore, the levels of oxidative stress, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß are all reduced significantly in PD mice and the CAR-25 mg kg-1 DA group in comparison with that in 6-OHDA-lesioned mice with saline and 6-OHDA-lesioned mice, as well as the Tyrosine hydroxylase-immunoreactive (TH-ir) density increased (p < 0.01). The toxicity of CAR was in vitro and in vivo investigated, showing that the safe dose of SWCNT-PEG exposure to PC12 cells was 6.25 µg µl-1 or lower with a higher metabolic activity in comparison with that in the control group and the safe dose of CAR in the mice experiments was 3.25 mg kg-1 or less, given by intraperitoneal injection with a lower level of oxidative stress and inflammatory responses in comparison with that in the control group. This study suggests that 25 mg kg-1 DA loaded onto 3.25 mg kg-1 CAR can alleviate the oxidative stress and inflammatory responses in parkinsonian mice and increase the TH-ir density in the striatum.

Gp85 genetic diversity of avian leukosis virus subgroup J among different individual chickens from a native flock.

To compare the genetic diversity and quasispecies evolution of avian leukosis virus (ALV) among different individuals, 5 chickens, raised in Shandong Provice of China, were randomly selected from a local chicken flock associated with serious tumor cases. Blood samples were collected and inoculated into chicken embryo fibroblast and DF-1 cell lines for virus isolation and identification, respectively, of Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and ALV. Five strains of ALV subgroup J (ALV-J) were identified, and the gp85 gene from each strain was amplified and cloned. For each strain, about 20 positive clones of gp85 gene were selected for sequence analyses and the variability of the quasispecies of the 5 strains was compared. The results showed that the nuclear acid length of gp85 gene of 5 ALV-J isolates is 921 bp, 921 bp, 924 bp, 918 bp, and 912 bp respectively, and amino acid homologies of different gp85 clones from the 5 ALV-J strains were 99.3 to 100%, 99.3 to 100%, 99.4 to 100%, 98.4 to 100%, 99.0 to 100%, respectively. The proportions of dominant quasispecies were 65.0%, 85.0%, 85.0%, 50.0%, 84.2%, respectively, and homology of the gp85 among these dominant quasispecies was 89.2 to 92.5%. These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock, and the dominant quasispecies continued to evolve both for their proportion and gene mutation.

A deep sequencing reveals significant diversity among dominant variants and evolutionary dynamics of avian leukosis viruses in two infectious ecosystems.

BACKGROUND: As a typical retrovirus, the evolution of Avian leukosis virus subgroup J (ALV-J) in different infectious ecosystems is not characterized, what we know is there are a cloud of diverse variants, namely quasispecies with considerable genetic diversity. This study is to explore the selection of infectious ecosystems on dominant variants and their evolutionary dynamics of ALV-J between DF1 cells and specific-pathogen-free (SPF) chickens. High-throughput sequencing platforms provide an approach for detecting quasispecies diversity more fully. RESULTS: An average of about 20,000 valid reads were obtained from two variable regions of gp85 gene and LTR-U3 region from each sample in different infectious ecosystems. The top 10 dominant variants among ALV-J from chicken plasmas, DF1 cells and liver tumor were completely different from each other. Also there was a difference of shannon entropy and global selection pressure values (ω) in different infectious ecosystems. In the plasmas of two chickens, a large portion of quasispecies contained a 3-peptides "LSD" repeat insertion that was only less than 0.01% in DF1 cell culture supernatants. In parallel studies, the LTR-U3 region of ALV-J from the chicken plasmas demonstrated more variants with mutations in their transcription regulatory elements than those from DF1 cells. CONCLUSIONS: Our data taken together suggest that the molecular epidemiology based on isolated ALV-J in cell culture may not represent the true evolution of virus in chicken flocks in the field. The biological significance of the "LSD" insert and mutations in LTR-U3 needs to be further studied.

A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice.

Wounds that fail to heal in a timely manner, for example, diabetic foot ulcers, pose a health, economic, and social problem worldwide. For decades, conventional wisdom has pointed to growth factors as the main driving force of wound healing; thus, growth factors have become the center of therapeutic developments. To date, becaplermin (recombinant human PDGF-BB) is the only US FDA-approved growth factor therapy, and it shows modest efficacy, is costly, and has the potential to cause cancer in patients. Other molecules that drive wound healing have therefore been sought. In this context, it has been noticed that wounds do not heal without the participation of secreted Hsp90α. Here, we report that a 115-aa fragment of secreted Hsp90α (F-5) acts as an unconventional wound healing agent in mice. Topical application of F-5 peptide promoted acute and diabetic wound closure in mice far more effectively than did PDGF-BB. The stronger effect of F-5 was due to 3 properties not held by conventional growth factors: its ability to recruit both epidermal and dermal cells; the fact that its ability to promote dermal cell migration was not inhibited by TGF-ß; and its ability to override the inhibitory effects of hyperglycemia on cell migration in diabetes. The discovery of F-5 challenges the long-standing paradigm of wound healing factors and reveals a potentially more effective and safer agent for healing acute and diabetic wounds.

[HIF-1α and neovascularization in early carotid atherosclerosis plaques in rabbits].

OBJECTIVE: To explore the relationship between hypoxia-inducible foctor-1α (HIF-1α) and neovascularization in early atherosclerosis plaques by establishing rabbit carotid atherosclerosis models, and to observe the value of contrast-enhanced ultrasound in the detection of neovascularization. METHODS: We provided high-fat diet combined with the implantation of silicone rubber ring to establish carotid atherosclerosis in rat models. On the 14th and 28th days, we detected neovascularization in the carotid atherosclerotic plaques by contrast-enhanced ultrasound, obtained the peak intensity (PI) of the contrast agent in the plaques by time-intensity curve (TIC) and analyzed the difference. We also tested the level of HIF-1α, vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), α-actin, and RAM-11 by immunohistochemical method in each group, analyzed their correlation, and the correlation between PI and CD31 expression. RESULTS: On the 14th day, contrast-enhanced ultrasound showed the neovascularization in the carotid atherosclerotic plaques. On the 14th and 28th days, the intensity of contrast-enhanced ultrasound showed significant difference, the mean optical density of HIF-1α, VEGF, CD31, RAM-11, and α-actin within the carotid atherosclerotic plaques also showed statistical difference. The expressions between HIF-1α and VEGF, HIF-1α and CD31, HIF-1α and RAM-11, HIF-1α and α-actin, as well as PI and CD31 showed highly positive correlations. CONCLUSION: During the process of atherosclerosis evolution, neovascularization in the atherosclerotic plaques has come into being in the early period, and HIF-1α in early atherosclerosis can promote the formation of neovascularization. Contrast-enhanced ultrasound can detect the dynamic changes of neovascularization within early atherosclerotic plaques.

Participation of the lipoprotein receptor LRP1 in hypoxia-HSP90alpha autocrine signaling to promote keratinocyte migration.

Hypoxia is a microenvironmental stress in many pathological conditions, including wound healing and tumor invasion. Under hypoxia, the cells are forced to adapt alternative and self-supporting mechanisms. Understanding these mechanisms may lead to new insights into human disorders. We report here a novel autocrine signaling mechanism by which hypoxia promotes human keratinocyte (HK) migration. First, hypoxia triggers HKs to secrete heat shock protein 90-alpha (HSP90alpha) via a HIF1-dependent pathway. The secreted HSP90alpha in turn promotes migration, but not proliferation, of the cells. Disruption of the secretion or extracellular function of HSP90alpha blocked hypoxia-stimulated HK migration. The ubiquitously expressed surface receptor, LRP1 (LDL-receptor-related protein 1), mediates the HSP90alpha signaling. Inhibition of LRP1 binding to extracellular HSP90alpha by neutralizing antibodies or genetic silencing of the LRP1 receptor by RNAi completely nullified hypoxia-driven HK migration. Finally, re-introducing a RNAi-resistant LRP1 cDNA into LRP1-downregulated HKs rescued the motogenic response of the cells to hypoxia. We propose that the hypoxia-HSP90alpha-LRP1 autocrine loop provides previously unrecognized therapeutic targets for human disorders such as chronic wounds and cancer invasion.

Non-compensating roles between Nckalpha and Nckbeta in PDGF-BB signaling to promote human dermal fibroblast migration.

Platelet-derived growth factor BB (PDGF-BB) is a Food and Drug Administration (FDA)-approved growth factor, acting as a mitogen and motogen of dermal fibroblasts (DFs), for skin wound healing. The two closely related SH2/SH3 adapter proteins, Nckalpha and Nckbeta, connect PDGF-BB signaling to the actin cytoskeleton and cell motility. The mechanism has not been fully understood. In this study, we investigated, side by side, the roles of Nckalpha and Nckbeta in PDGF-BB-stimulated DF migration. We found that cells expressing the PDGFRbeta-Y751F mutant (preventing Nckalpha binding) or PDGFRbeta-Y1009F mutant (preventing Nckbeta binding), DF cells isolated from Nckalpha- or Nckbeta-knockout mice, and primary human DF cells with RNA interference (RNAi) knockdown of the endogenous Nckalpha or Nckbeta all failed to migrate in response to PDGF-BB. Overexpression of the middle SH3 domain of Nckalpha or Nckbeta alone in human DFs also blocked PDGF-BB-induced cell migration. However, neither Nckalpha nor Nckbeta was required for the activation of the PDGF receptor, p21-activated protein kinase (Pak1), AKT, extracellular signal-regulated kinase (ERK) 1/2, or p38MAP by PDGF-BB. Although PDGF-BB stimulated the membrane translocation of both Nckalpha and Nckbeta, Nckalpha appeared to mediate Cdc42 signaling for filopodium formation, whereas Nckbeta mediated Rho signaling to induce stress fibers. Thus, this study has elucidated the independent roles and mechanisms of action of Nckalpha and Nckbeta in DF migration, which is critical for wound healing.

PKCdelta clustering at the leading edge and mediating growth factor-enhanced, but not ecm-initiated, dermal fibroblast migration.

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cdelta (PKCdelta). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCdelta is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCdelta signaling with dominant-negative kinase-defective PKC (PKCdelta-KD) or PKCdelta-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCdelta-KD nor PKCdelta-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCdelta (PKCdelta-R144/145A) partially mimics the effect of PDGF-BB. However, PKCdelta-KD, PKCdelta-siRNA, or PKCdelta-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCdelta blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCdelta in the control of DF migration.

Mechanism of human dermal fibroblast migration driven by type I collagen and platelet-derived growth factor-BB.

Migration of human dermal fibroblasts (HDFs) is critical for skin wound healing. The mechanism remains unclear. We report here that platelet-derived growth factor-BB (PDGF-BB) is the major promotility factor in human serum for HDF motility on type I collagen. PDGF-BB recapitulates the full promotility activity of human serum and anti-PDGF neutralizing antibodies completely block it. Although collagen matrix initiates HDF migration without growth factors, PDGF-BB-stimulated migration depends upon attachment of the cells to a collagen matrix. The PDGF-BB's role is to provide directionality and further enhancement for the collagen-initiated HDF motility. To study the collagen and PDGF-BB "dual signaling" in primary HDF, we establish "gene cassettes" plus lentiviral gene delivery approach, in which groups of genes are studied individually or in combination for their roles in HDF migration. Focal adhesion kinase, p21(Rac,CDC42)-activated kinase and Akt are grouped into an upstream kinase gene cassette, and the four major mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 5) are grouped into a downstream kinase gene cassette. The experiments demonstrate 1) the genes' individual roles and specificities, 2) their combined effects and sufficiency, and 3) the mechanisms of their intermolecular connections in HDF migration driven by collagen and PDGF-BB.